Protocols for Co-Culture Phenotypic Assays with Breast Cancer Cells and THP-1-Derived Macrophages.

Tumor mass comprises not only cancer cells but also heterogeneous populations of immune and stromal cells, along with the components of the extracellular matrix, collectively called the tumor microenvironment (TME). This diverse population of cells can communicate with each other, which can positively or negatively affect tumor growth and progression to malignancy. The most common type of immune cells in the TME are macrophages. Macrophages continuously differentiate into a broad landscape of tumor-associated macrophages (TAMs) in response to numerous signals from the TME, which makes studies on TAMs quite challenging. Therefore, implementing reliable protocols is a milestone for drawing consistent conclusions about the interactions between cancer cells and TAMs. Here, we provide the details for the polarization of a human leukemia monocytic cell line, THP-1, into M0, M1 and M2 macrophages. We also present a step-by-step protocol for a transwell co-culture using a human breast cancer cell line, HCC1806, and THP-1-derived macrophages. Finally, we describe the colony formation and migration assays performed on the breast cancer cells after the co-culture with macrophages to measure the influence of macrophages on the oncogenic features of cancer cells. In summary, our co-culture-based protocols can be a valuable resource for investigating the interactions between macrophages and cancer cells.

MLK4 regulates DNA damage response and promotes triple-negative breast cancer chemoresistance.

Chemoresistance constitutes a major challenge in the treatment of triple-negative breast cancer (TNBC). Mixed-Lineage Kinase 4 (MLK4) is frequently amplified or overexpressed in TNBC where it facilitates the aggressive growth and migratory potential of breast cancer cells. However, the functional role of MLK4 in resistance to chemotherapy has not been investigated so far. Here, we demonstrate that MLK4 promotes TNBC chemoresistance by regulating the pro-survival response to DNA-damaging therapies. We observed that MLK4 knock-down or inhibition sensitized TNBC cell lines to chemotherapeutic agents in vitro. Similarly, MLK4-deficient cells displayed enhanced sensitivity towards doxorubicin treatment in vivo. MLK4 silencing induced persistent DNA damage accumulation and apoptosis in TNBC cells upon treatment with chemotherapeutics. Using phosphoproteomic profiling and reporter assays, we demonstrated that loss of MLK4 reduced phosphorylation of key DNA damage response factors, including ATM and CHK2, and compromised DNA repair via non-homologous end-joining pathway. Moreover, our mRNA-seq analysis revealed that MLK4 is required for DNA damage-induced expression of several NF-кB-associated cytokines, which facilitate TNBC cells survival. Lastly, we found that high MLK4 expression is associated with worse overall survival of TNBC patients receiving anthracycline-based neoadjuvant chemotherapy. Collectively, these results identify a novel function of MLK4 in the regulation of DNA damage response signaling and indicate that inhibition of this kinase could be an effective strategy to overcome TNBC chemoresistance.